A neurotoxin (NT) produced by the ubiquitous, spore forming bacteria Clostridium botulinum causes the neuroparalytic disease botulism. The disease generally results from ingestion of NT (food poisoning). A puzzling toxico-infectious form of the disease, not caused by ingestion of the NT and thought to be confined to 3-35 week old humans (infant botulism), was recently confirmed in adults. The NT blocks release of the neurotransmitter acetylcholine (ACh) at neuromuscular junctions inducing flaccid muscle paralysis that can lead to death. The mechanism of action of the NT is not known in detail other than that it acts in 3 steps. The dichain NT molecule appears to have 3 functional domains for extracellular binding, channel formation and blocking of ACh release. Little is known about the antigenic characteristics of the NT except that it is found in at least 7 antigenically distinct forms. And yet, definitive diagnosis and therapy depends on the antiserum raised with toxoids that are approximately 90% impure preparations of NT detoxified with formalin. No drug is effective for therapy of botulism other than the sero-type specific antiserum that neutralizes the unbound NT circulating in the host. As part of long-term on-going studies of the structure, structure- function relationship, and mode of action of the NT we need to i) determine the sequence of the entire protein in order to map (with site specific probes) its various regions that are critical for ii) neurotoxicity (binding, internalization and blockage of ACh release), iii) eliciting antibodies and iv) antibody binding. We plan to 1) determine the entire amino acid sequence of this large protein (Mr150,000) by the most efficient technique known-- nucleotide sequencing of its gene. 2) Monoclonal antibodies (MAB) will be produced and characterized for different binding sites on the NT. MABs will be tested for their ability to inactivate the three functional domains by binding at or close to the active site. 3) Fine structure of the antibody binding sites will be examined by specific chemical modification of amino acid residues. 4) Second generation toxoids will be developed from approximately 99% pure NT following chemical modification with least damage to its serological reactivity. As immunogens, these toxoids will be significantly superior to the ones available now.